Synthesis of Mixed Opioid Affinity Cyclic Endomorphin-2 Analogues with Fluorinated Phenylalanines

Abstract

As part of our continuing studies on the structure−activity relationships of cyclic pentapeptides based on the structure of endomorphin-2 (EM-2), we report here the synthesis and biological activities of a new series of analogues of a general sequence Tyr/Dmt-c[D-Lys-Phe-Phe-Asp]NH 2 (where Dmt = 2′,6′-dimethyltyrosine), incorporating fluorinated amino acids: 4-fluorophenylalanine (4-F-Phe), 2,4-difluorophenylalanine (2,4-F-Phe), or 4-trifluoromethylphenylalanine (4-CF 3 -Phe) instead of the Phe residue in position 3 or 4. Depending on the fluorinated amino acid residue and its position in the sequence, analogues were mixed, high affinity MOP/KOP receptor agonists, MOP/DOP/KOP agonists, or selective KOP agonists. The in vitro potencies and efficacies of all novel analogues were assessed in calcium mobilization assay. The most potent analogues, Dmt-c[D-Lys-Phe-4-F-Phe-Asp]NH 2 and Dmt-c[D-Lys-Phe-2,4-F-Phe-Asp]NH 2 , were tested in vivo in the mouse hot-plate test. They produced strong antinociceptive effect not only after intracerebroventricular but also after intraperitoneal injection, indicating that they were able to cross the blood−brain barrier.

Pharmacological Profile and Molecular Modeling of Cyclic Opioid Analogues Incorporating Various Phenylalanine Derivatives

“…On the other hand, in the case of smaller substituents (pF, 2,4-diF, oMe, mMe, pMe), the IC 50 values increased only to some extent and remain in low nanomolar range. [35,36] A more pronounced deterioration occurred with shortening of the side chain, as in derivatives 3 and 9. Thus, there exists a qualitative, parabolic relationship between the μOR affinity and the volume of the side chain in the third position ( Figure 7).…”

“…Given the hydrophobic character of the interactions at the S2 and S3 sites, it could be further expected that smaller side chains should also bring about affinity decreases, as the result of underpacking of the site. [34] Indeed, derivatives with pCF 3 substitution at Phe3 ring ( [35] ) exhibited a significantly decreased affinity for μOR with IC 50 values being greater than 1000 nM. On the other hand, in the case of smaller substituents (pF, 2,4-diF, oMe, mMe, pMe), the IC 50 values increased only to some extent and remain in low nanomolar range.…”

Cyclic Opioid Peptides

“…The incorporation of DDap 2 , affording a 14-membered ring, resulted in a highly selective MOR ligand with high affinity in the picomolar range. Another 14-membered cyclic analogue, Tyr-c 2,4 [DLys-Phe-Asp]-NH 2 , gave a similar biological profile to the DDap 2 -analogue, suggesting the optimal ring size for MOR affinity and selectivity to be 14.The cyclic EM-2 analogue Tyr/Dmt-c 2,5 [DLys-Xxx-Yyy-Asp]-NH 2 was also modified at position 3 or 4 using Phe, Phe(4-F), Phe(2,4,-difluoro), or Phe(4-CF 3 ) [49]. All compounds exhibited picomolar affinities at the MOR with the exception of the Phe(4-CF 3 ) 3 containing analogues which only interact with the KOR.…”

Fine‐Tuning the Proteolytic Stability of Peptides with Fluorinated Amino Acids

“…In 2015, the synthesis and biological activity of fluorinated, cyclic pentapeptides based on the structure of endomorphin‐2 (EM‐2) with the general sequence Tyr/Dmt‐c[ d ‐Lys‐Phe‐Phe‐Asp]NH 2 (Dmt = 2′,6′‐dimethyltyrosine) was reported . Endomorphins have attracted much attention as useful drugs for pain relief, but their application is limited by their relatively poor receptor selectivity, rapid degradation in vivo, insufficiency in crossing the blood‐brain barrier, as well as toxic side effects .…”

“…Endomorphins have attracted much attention as useful drugs for pain relief, but their application is limited by their relatively poor receptor selectivity, rapid degradation in vivo, insufficiency in crossing the blood‐brain barrier, as well as toxic side effects . Thus, the Phe residue in either position 3 or 4 of the EM‐2 peptide was individually substituted with 4‐fluorophenylalanine (4‐F‐Phe), 2,4‐difluorophenylalanine (2,4‐F 2 ‐Phe) or 4‐trifluoromethylphenylalanine (4‐CF 3 ‐Phe) (Figure ) . Stability towards enzymatic digestion was determined in vitro by incubation with rat brain homogenate for 90 min.…”